Antibodies against heart muscle actin

ABSTRACT

The present invention relates to antibodies against heart muscle actin, processes for the production of such antibodies and their use.

This application is a continuation of PCT/DE96/00620 filed Apr. 9, 1996.

The present invention relates to antibodies against heart muscle actin,processes for the production thereof and their use.

Processes are known for detecting a cardiac infarct by determining theheart muscle-specific creatine phosphokinase and by determining theserum-lactate dehydrogenase, respectively. However, the formerdetermination cannot be made until 3 hours following a cardiac infarct.Also, only a maximum of 50% of the creatine phosphokinase is presentafter one day. Furthermore, the serum-lactate dehydrogenase is not aheart-specific enzyme. Thus, the above determinations are only suitablein a limited fashion for detecting a cardiac infarct or another lesionof the heart muscle.

Therefore, it is the object of the present invention to provide aproduct by which a cardiac infarct or another lesion of the heart musclecan be detected specifically and reliably.

According to the invention this is achieved by antibodies which aredirected against heart muscle actin.

It follows from applicant's experiments that in the case of cardiacinfarct heart muscle actin is released. This actin differs as regardsfew amino acids from the actin of the sceletal muscle and that of thesmooth muscles, respectively (cf. Vandekerckhove, J. and Weber, K.,Differentiation 14 (1979), pp. 123-133). According to the inventionthese differences are used to direct antibodies against heart muscleactin.

Such antibodies may be polyclonal or monoclonal, monoclonal antibodiesbeing preferred. The antibodies may have been obtained from any animalor human being, rabbits being preferred for polyclonal antibodies andmice being preferred for monoclonal antibodies.

In addition, the antibodies may be synthetic, portions which are notnecessary for detecting heart muscle actin being optionally lackingfully or partially therefrom and these portions being replaced by otherswhich provide the antibodies with further favorable properties,respectively.

The expression "heart muscle actin" comprises heart muscle actin of anykind and origin as well as fragments thereof. It may originate fromhuman beings or animals. In addition, the heart muscle actin may bepresent in free or complexed form.

Preferred antibodies of the present invention, namely the monoclonalmouse antibodies Ac1-12.3.1 and Ac1-20.4.2 were deposited with DSMZlocated at Mascheroder Weg 1b 38124 Braunschweig, Germany [German-TypeCollection of Microorganisms] under numbers DSM ACC 2208 and DSM ACC2209, respectively, on Mar. 22, 1995 in accordance with the terms of theBudapest Treaty.

Antibodies according to the invention may be produced according toconventional processes. If polyclonal antibodies and monoclonalantibodies, respectively, shall be produced, it will be favorable toimmunize animals, particularly rabbits for the former antibodies andmice for the latter antibodies, with an above heart muscle actin and/orfragments thereof, particularly fragments of the N-terminus orC-terminus of heart muscle actin. The expression "fragments thereof"also comprises synthetic peptides which include partial sequences,particularly of the N-terminus or C-terminus, of heart muscle actin. Itmay also be advantageous to immunize the animals with a mixtureconsisting of heart muscle actin and/or fragments thereof. Furtherboosters of the animals may take place with the same heart muscle actinor actins and/or fragments thereof. It is also possible to use otherheart muscle actins and/or fragments thereof or a combination of theseand the preceding heart muscle actin or actins and/or fragments thereoffor booster.

The polyclonal antibodies may then be obtained from the serum of theanimals. Spleen cells from the animals are fused with myeloma cells forthe monoclonal antibodies.

For the production of synthetic antibodies, e.g. the above obtainedmonoclonal antibodies may be used as a basis. For this purpose, it is anobvious thing to analyze the antigen binding regions of the monoclonalantibodies and identify the portions necessary and not necessary for thespecific heart muscle actin detection. The necessary portions may thenbe modified and the unnecessary portions may be fully or partiallyeliminated and be replaced by portions which provide the antibodies withfurther favorable properties, respectively. Portions can also bemodified, eliminated or replaced outside the binding regions of theantibodies. A person skilled in the art is familiar with the fact thatparticularly the DNA recombination technology is suited for the abovemeasures. He is perfectly familiar with this fact.

Antibodies according to the invention distinguish themselves in thatthey recognize specifically heart muscle actin and fragments thereof,respectively. Therefore, the antibodies are suitable for a rapid andreliable detection of heart diseases in connection with which heartmuscle actin is released. These diseases comprise particularly thecardiac infarct.

The above diseases may be detected by any detection methods,particularly a Western blot, an ELISA, an immunoprecipitation or byimmunofluorescence. For this purpose, the antibodies according to theinvention may be labeled, if appropriate, or be used in combination withlabeled antibodies directed thereagainst. Moreover, the above antibodiesmay be used in a biosensor process.

According to the invention kits are also provided which contain theabove antibodies together with carrier materials and conventionalauxiliary agents such as buffers.

BRIEF DESCRIPTION OF THE DRAWING

The figure shows a Western blot in which antibodies according to theinvention recognize specifically heart muscle actin over skeletal muscleactin. The respective position of actin is shown on the right-handmargin of the figure by a large arrow each. Lanes 7 and 11,respectively, from (a) show the reaction with heart muscle actin andlanes 7 and 11, respectively, from (b) show the non-reaction withskeletal muscle actin.

The present invention is explained by the below examples.

EXAMPLE 1 Production of Monoclonal Antibodies Against Heart Muscle Actin

Mice of the Balb/c strain were used for immunization. An N-terminaldecapeptide of heart muscle actin was used as antigen. This decapeptidecontains the following amino acid sequence: DDEETTALVC (SEQ ID No:1).Its N-terminus has an acetyl group, and its C-terminus includes an amidegroup. The decapeptide was linked as usual with bovine serum albumin(BSA) and Keyhole Limpet Hemocyanin (KLH), respectively.

Immunization and Booster Pattern

100 μg of decapeptide conjugated with BSA emulsified in completeFreund's adjuvant were administered to a mouse.

This was followed by three booster injections using in each case 100 μgdecapeptide conjugated in change with KLH (twice) and BSA (once),respectively, the decapeptide having been emulsified in incompleteFreund's adjuvant.

Four days prior to the taking of the spleen cells, the mouse was givenintraperitoneally 100 μg of decapeptide conjugated with BSA.

The removed spleen cells were fused with mouse myeloma cells of theknown strain X63-Ag8, 653.

Monoclonal antibodies were obtained. Of these, the antibodies referredto as Ac1-12.3.1 and Ac1-20.4.2, respectively, were deposited with DSMunder numbers DSM ACC 2208 and DSM ACC 2209, respectively, on Mar. 22,1995.

EXAMPLE 2 Detection of Heart Muscle Actin by Antibodies According to theInvention

Bovine heart and rabbit muscle were used for the production of proteinpreparations each. This production was carried out according to aconventional process. The protein preparations were subjected to apolyacrylamide gel electrophoresis and the polypeptides separated inthis way were transferred to a nitrocellulose membrane. The latter wasthen incubated with the above antibodies ACC 2208 and ACC 2209, dilutedat 1:10 and 1:50, respectively, at 37° C. for 1 hour. After several washsteps using PBS (0.05% Tween 20) and optionally a wash step using 0.5 MNaCl in PBS, a purchasable anti-mouse antibody coupled to alkalinephosphatase (dilution according to the manufacturer's instructions) wasadded. After 30 minutes of incubation at 37° C., several wash stepsusing PBS were carried out and thereafter the alkaline phosphatasedetection reaction with developer solution (36 μM5'-bromo-4-chloro-3-indolylphosphate, 400 μM nitro blue tetrazolium, 100mM Tris-HCl, pH 9.5, 100 mM NaCl, 5 mM MgCl₂) followed at roomtemperature until bands were visible.

It turned out that the antibodies ACC 2008 and ACC 2209 according to theinvention recognize specifically heart muscle actin over skeletal muscleactin. In this connection, the degree of specificity of the antibodiesis especially high when the above wash step is carried out with 0.5 mNaCl in PBS.

    __________________________________________________________________________    #             SEQUENCE LISTING                                                   - -  - - <160> NUMBER OF SEQ ID NOS: 1                                        - - <210> SEQ ID NO 1                                                        <211> LENGTH: 10                                                              <212> TYPE: PRT                                                               <213> ORGANISM: Artificial Sequence                                           <220> FEATURE:                                                                <223> OTHER INFORMATION: Description of Artificial - #Sequence:             n-terminal                                                                            peptide from heart muscle actin                                          - - <400> SEQUENCE: 1                                                         - - Asp Asp Glu Glu Thr Thr Ala Leu Val Cys                                    1               5 - #                 10                                  __________________________________________________________________________

What is claimed:
 1. An anti-heart muscle actin antibody which binds toheart muscle actin but not skeletal muscle, wherein antigen specificityis determined after a 0.5 M NaCl wash.
 2. The antibody of claim 1,wherein said antibody is polyclonal.
 3. The antibody of claim 1, whereinsaid antibody is monoclonal.
 4. The antibody according to claim 3,wherein said antibody is deposited with DSM [German-Type CultureCollection of Microorganisms] under ACC
 2208. 5. The antibody accordingto claim 3, wherein said antibody is deposited with DSM under ACC 2209.6. A process for the production of an antibody according to claim 1,comprising(1) immunizing an animal with a peptide consisting of SEQ IDNO:1, and (2a) obtaining polyclonal antibodies from the serum of theanimal, or (2b) obtaining monoclonal antibodies after fusion of spleencells from the animal with myeloma cells,such that an anti-heart muscleactin antibody which binds to heart muscle actin but not skeletal muscleis produced.
 7. A method for lesions of heart muscle, said methodcomprising contacting a biological fluid sample with the antibody ofclaim 1 such that the presence or absence of heart muscle actin orfragments thereof is detected.
 8. The method according to claim 7,wherein the lesion is the result of a cardiac infarct.
 9. The methodaccording to claim 7, wherein said method involves a detection stepselected from the group consisting of Western blot, an ELISA, animmunofluorescence method, an immunoprecipitation and a biosensorprocess.